Bridging Project 4: Haloacid Dehalogenase (HAD) Superfamily

HAD超家族是磷酸转移酶（磷酸变位酶，ATP酶和磷酸酶）的一个大家族（约19，000个非冗余序列）[1]，在生命的所有三个王国[2-4]中都有代表，并且在每个细胞中，有大量的同源物（E.大肠杆菌35株，鼠伤寒沙门氏菌35株，铜绿假单胞菌31株，结核分枝杆菌30株，蜡样芽孢杆菌31株，脆弱类杆菌24株， 肺炎链球菌中24个;酿酒酵母中45个;秀丽隐杆线虫中84个;拟南芥中169个;莫氏卷柏中292个;人中183个）。多达80-90%的成员是磷酸酶[5]，其中绝大多数功能未知。 大约40%的细菌代谢物组由磷酸化代谢物组成[6]。 磷酸盐取代基是常见的，因为它们增强了代谢物的水溶性以及其以高亲和力和特异性结合代谢酶的能力。从磷酸化代谢物中除去磷酸基团是通过磷酸酶进行的。特定磷酸酶的“功能”是 由其在细胞中靶向的磷酸化代谢物定义，即，被它的“生理基质”。因此，HAD磷酸酶满足涉及磷酸化大分子和代谢物的细胞过程和代谢途径的需求。HAD磷酸酶功能的差异是基于底物识别元件的差异。底物识别元件与位于核心结构域中的催化支架分离（图1A）。 形成活性位点的四个肽段或“基序”定位于保守的/艾德化的Asp亲核体、Asp酸/碱、Lys/Arg和Ser/Thr磷酸结合残基以及Mg^* 辅因子Asp/Glu结合残基（图1B）。这些残基与骨架主链元件结合，形成稳定三角双锥过渡态/中间体的空间和静电模 在反应过程中产生的（图1B）[7]。HAD磷酸酶底物识别元件位于通过溶剂化接头栓系到核心结构域的帽结构域（如在HAD C1和C2类中，也称为I型和11型）中，或位于从核心结构域延伸的短环/螺旋片段中（如在“无帽”HAD C 0类中，也称为III型）（图1A）[8]。 虽然HAD磷酸酶具有相同的催化位点并通过相同的第二部分反应进行，但它们能够利用第一部分反应的底物结合步骤和随后的加成-消除步骤的特定结构要求来区分生理底物和其他磷酸化物质（大分子和代谢物）。其中底物结合之后是帽结构域或环闭合的诱导拟合模型适用于大多数HAD磷酸酶。 底物离去基团和帽结构域/加芬环之间的有利静电相互作用将有助于底物结合亲和力。为了有效的周转，磷酰基必须在催化位点内以正确的方向结合。如果底物离去基团太大或太小， 可能发生非生产性绑定。因此，从催化位点延伸到活性位点入口的acfive位点区域的大小、形状和静电表面可以提供对生理底物的身份的重要洞察。这用作使用虚拟筛选（通过结构核心和计算核心成为可能）来识别本文中的生理底物的候选物的基础。 由实验活性筛选定义的底物特异性表明，典型的HAD磷酸酶具有松散的底物特异性和适度的催化效率。因此，单独的活性筛选通常不能验证实际的生理底物。相反，他们提供的候选人可以使用序列/基因组分析核心和微生物学核心提供的工具进一步询问。

The HAD superfamily is a large enzyme family (~19,000 nonredundant sequences) [1] of phosphotransferases (phosphomutases, ATPases and phosphatases) represented in all three kingdoms of life [2-4], and, within each cell, by a large number of homologs (28 in E. coli; 35 in Salmonella typhimurium; 31 in Pseudomonas aeruginosa; 30 in Mycobacterium tuberculosis; 31 in Bacillus cereus; 24 in Bacteroides fragilis; 24 in Streptococcus pneumoniae; 45 in Saccharomyces cerevisiae; 84 in Caenorhabditis elegans; 169 in Arabidopsis thaliana; 292 in Selaginella moeltendorffii; 183 in human). As many as 80-90% of the members are phosphatases [5], the vast majority of which have unknown functions. Approximately 40% of the bacterial metabolome is comprised of phosphorylated metabolites [6]. Phosphate substituents are common because they enhance the water solubility of the metabolite as well as its ability to bind to metabolic enzymes with high affinity and specificity. The removal of phosphate groups from phosphorylated metabolites is performed by phosphatases. The "function" of a particular phosphatase is defined by the phosphorylated metabolite that it targets in the cell, i.e., by its "physiological substrate". Thus, the HAD phosphatases meet the demands of cellular processes and metabolic pathways that involve phosphorylated macromolecules and metabolites. Divergence in HAD phosphatase funcfion is based on the divergence of the substrate-recognition elements. The substrate-recognition elements are separate from the catalytic scaffold, which is located in the core domain (Figure 1A). The four pepfide segments or "motifs" which form the active site position the consen/ed Asp nucleophile, Asp acid/base, the Lys/Arg and Ser/Thr phosphate-binding residues and the Mg^* cofactor Asp/Glu binding residues (Figure IB). These residues, in combinafion with the scaffold main-chain elements, form a steric and electrostafic mold that stabilizes the trigonal bipyramidal transifion states/intermediates produced along the reaction pathway (Figure 1B) [7]. The HAD phosphatase substrate recognition elements are located in either a cap domain (as in HAD classes Cl and C2, also known as Type I and Type 11) tethered to the core domain by a solvated linker, or in short loop/helical segments that extend from the core domain (as in the "capless" HAD class CO also known as Type III) (Figure 1A) [8]. Although HAD phosphatases possess the same catalytic site and proceed through the same second partial reaction, they are able to use the specific structural requirements of the substrate-binding step and the subsequent addition-eliminafion steps of the first partial reacfion to discriminate between the physiological substrate and other phosphorylated species (macromolecules and metabolites). The induced fit model, wherein substrate binding is followed by cap domain or loop closure, applies to most HAD phosphatases. Favorable electrostafic interaction between the substrate leaving group and the cap domain/gafing loops will contribute to the substrate-binding affinity. For efficient turnover, the phosphoryl group must be bound in the correct orientation within the catalyfic site. If the substrate-leaving group is too large or too small, nonproductive binding is likely to occur. Thus, the size, shape and electrostafic surface ofthe acfive site region that extends from the catalytic site to the active site entrance can provide significant insight into the identity of the physiological substrate. This serves as the basis for the use of virtual screening (made possible by the Structure Core and Computation Core) to identify candidates for the physiological substrate herein. Substrate specificities defined by experimental activity screens suggest that the typical HAD phosphatase has loose substrate specificity coupled with modest catalytic efficiency. Thus, acfivity screens alone often cannot idenfify the actual physiological substrate. Rather, they provide candidates that can be further interrogated using the tools provided by the Sequence/Genome Analysis Core and Microbiology Core.